Phospholipid scramblase 1 (PLSCR1) is a Ca2+-binding, endofacial, plasma membrane protein that was originally proposed to contribute to the transbilayer movement of phosphatidylserine and other membrane phospholipids (PL) that is observed upon platelet activation, or with cell injury and apoptosis. We recently discovered that expression of PLSCR1 is transcriptionally induced by several cytokines, including certain growth factors that are known to regulate proliferation, differentiation, and mobilization of blood leukocytes. Furthermore, we found that PLSCR1 is itself a substrate of protein kinases that participate in signaling by these same cytokine-activated receptors. A spontaneous mutation identified in murine PLSCRI that deleted N-terminal codons was reported to confer leukemogenic potential, and we observed that mice with targeted disruption of the PLSCR1 locus exhibit delayed fetal production of mature blood granulocytes and defective granulocytosis in response to cytokine stimulation. When taken together, these data suggest that PLSCR1, and potentially other recently identified members of this gene family, play a previously unrecognized role in growth factor receptor-initiated pathways that regulate leukocyte differentiation and proliferation from precursor cells. By contrast to the observed hematopoietic defects, PLSCR1-/- animals show no hemostatic abnormality and plasma membrane PL scramblase activity appears normal, although this may reflect redundant contribution of another member of the PLSCR gene family (PLSCR3) that is also widely-expressed in various blood cells. During the next five years of this Project, our overall goals are to (1) definitively resolve whether the PLSCR proteins mediate the PL scramblase activity of the plasma membrane, and, (2) elucidate the cellular and molecular mechanisms by which the PLSCR proteins influence cytokine-regulated maturation, proliferation, and potentially function of mature blood leukocytes. Our specific aims include: AIM 1. To evaluate plasma membrane PL scramblase activity of blood cells from mice deficient in both PLSCR1 & PLSCR3; AIM 2. To identify and characterize abnormalities of leukocyte production and leukocyte function in PLSCR1-/- and PLSCR3-/- mice; and AIM 3. To determine how PLSCR1 (and potentially, PLSCR3) functions in signaling and/or effector pathways initiated through c-kit, G-CSFR, and Fcepsilon-R1. We believe that the results of these experiments will substantially advance our understanding of the biologic function of this unique gene family and will reveal new regulatory mechanisms that influence myeloid differentiation, proliferation and function.